Maize metallothionein gene and promoter

ABSTRACT

The present invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions include a novel nucleotide sequence for a root-preferred promoter for the gene encoding, a metallothionein gene and sequences isolated therefrom. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises transforming a plant cell with a nucleotide sequence operably linked to one of the root-preferred promoters of the present invention and regenerating a stably transformed plant that expresses the nucleotide sequence in a root-preferred manner from the transformed plant cell. Compositions and methods for expressing metallothionein genes in plants, plant cells and tissues are also provided. The compositions comprise nucleotide sequences encoding plant metallothionein. The sequences are useful in transforming plants for tissue-preferred or constitutive expression of metallothionein. Such sequences find use in modulating levels of metal ions in plants and plant tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/520,268, filed Mar. 7, 2000, which claims the benefit of U.S.Provisional Application No. 60/123,510, filed Mar. 8, 1999, which arehereby incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of plant molecularbiology, more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

[0003] Expression of heterologous DNA sequences in a plant host isdependent upon the presence of an operably linked promoter that isfunctional within the plant host. Choice of the promoter sequence willdetermine when and where within the organism the heterologous DNAsequence is expressed. Where expression in specific tissues or organs isdesired, tissue-preferred promoters may be used. Where gene expressionin response to a stimulus is desired, inducible promoters are theregulatory element of choice. In contrast, where continuous expressionis desired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in the expressionconstructs of transformation vectors to bring about varying levels ofexpression of heterologous nucleotide sequences in a transgenic plant.

[0004] Frequently it is desirable to express a DNA sequence inparticular tissues or organs of a plant. For example, increasedresistance of a plant to infection by soil- and air-borne pathogensmight be accomplished by genetic manipulation of the plant's genome tocomprise a tissue-preferred promoter operably linked to a heterologouspathogen-resistance gene such that pathogen-resistance proteins areproduced in the desired plant tissue.

[0005] Alternatively, it might be desirable to inhibit expression of anative DNA sequence within a plant's tissues to achieve a desiredphenotype. In this case, such inhibition might be accomplished withtransformation of the plant to comprise a tissue-preferred promoteroperably linked to an antisense nucleotide sequence, such thatexpression of the antisense sequence produces an RNA transcript thatinterferes with translation of the mRNA of the native DNA sequence.

[0006] Thus far, the regulation of gene expression in plant roots hasnot been adequately studied despite the root's importance to plantdevelopment. To some degree this is attributable to a lack of readilyavailable, root-specific biochemical functions whose genes may becloned, studied, and manipulated. Genetically altering plants throughthe use of genetic engineering techniques and thus producing a plantwith useful traits requires the availability of a variety of promoters.An accumulation of promoters would enable the investigator to designrecombinant DNA molecules that are capable of being expressed at desiredlevels and cellular locales. Therefore, a collection of tissue-preferredpromoters would allow for a new trait to be expressed in the desiredtissue. Several genes have been described by Takahashi et al. (1991),Plant J. 1:327-332; Takahashi et al. (1990), Proc. Natl. Acad. Sci. USA87:8013-8016; Hertig et al. (1991), Plant Mol Biol. 16:171-174; Xu etal. (1995), Plant Mol Biol. 27:237-248; Capone et al. (1994), Plant MolBiol. 25:681-691; Masuda et al. (1999), Plant Cell Physiol.40(11):1177-81; Luschnig et al. (1998), Genes Dev. 12(14):2175-87;Goddemeier et al. (1998), Plant Mol Biol. 36(5):799-802; and Yamamoto etal. (1991), Plant Cell. 3(4):371-82 to express preferentially in plantroot tissues.

[0007] Metallothioneins (MT's) are proteins or polypeptides that bindand sequester ionic forms of certain metals in plant and animal tissues.Examples of such metals include copper, zinc, cadmium, mercury, gold,silver, cobalt, nickel and bismuth. The specific metals sequestered byMT's vary for the structurally distinct proteins/polypeptides occurringin different organisms. Plants appear to contain a diversity ofmetal-binding MT's with the potential to perform distinct roles in themetabolism of different metal ions. In plants, MT's are suggested tohave roles in metal accumulation, metal intoxication, and embryogenesis.

[0008] Typically, MT's and MT-like proteins are Cys-rich proteins,characterized by the presence of Cys-Xaa-Cys motifs suggesting thecapability of binding metal ions. Further categories of MT-like proteinshave been proposed on the basis of the predicted locations of Cysresidues and designated types 1 and 2. In type 1 there are exclusivelyCys-Xaa-Cys motifs, whereas in type 2 there is a Cys-Cys and aCys-Xaa-Xaa-Cys pair within the N-terminal domain. The type 1 motif hasbeen implicated in the binding and sequestration of copper.

[0009] Several metallothionein-like plant genes have been isolated,including those from pea, maize, barley, mimulus, soybean, castorbeanand arabidopsis. See Robinson et al. (1993) Biochem J. 295: 1-10.Sequences expressed in roots have been reported to be isolated from pea,as described in Evans et al. (1990) FEBS Lett 262:29-32. A maize root MTgene has been described in U.S. Pat. No. 5,466,785; though this sequenceis also expressed leaves, pith and seed, as described in de Framond(1991) FEBS Lett 290:103-106.

[0010] Thus, isolation and characterization of tissue-preferred,particularly root-preferred, promoters that can serve as regulatoryregions for expression of heterologous nucleotide sequences of interestin a tissue-preferred manner are needed for genetic manipulation ofplants. Furthermore, isolation and characterization of sequencesinvolved in metal-binding and accumulation are needed for influencingmetabolism of metals in plants.

SUMMARY OF THE INVENTION

[0011] Compositions and methods for regulating expression ofheterologous nucleotide sequences in a plant are provided. Compositionscomprise novel nucleotide sequences for promoters that initiatetranscription in a root-preferred manner. More particularly, atranscriptional initiation region isolated from a plant metallothioneingene, is provided. A method for expressing a heterologous nucleotidesequence in a plant using the transcriptional initiation sequencesdisclosed herein is provided. The method comprises transforming a plantcell with a transformation vector that comprises a heterologousnucleotide sequence operably linked to one of the plant promoters of thepresent invention and regenerating a stably transformed plant from thetransformed plant cell. In this manner, the promoter sequences areuseful for controlling the expression of operably linked codingsequences in a root-preferred manner.

[0012] Downstream from and under the transcriptional initiationregulation of the promoter will be a sequence of interest that willprovide for modification of the phenotype of the plant. Suchmodification includes modulating the production of an endogenousproduct, as to amount, relative distribution, or the like, or productionof an exogenous expression product to provide for a novel function orproduct in the plant.

[0013] Also provided are compositions and methods for expressingmetallothionein genes in plants, plant cells, and plant tissues. Thecompositions comprise nucleotide sequences for the expressed region ofthe MT gene, which comprise the nucleotide sequences encoding themetallothionein polypeptide. These sequences are useful in transformingplants for tissue-preferred or constitutive expression ofmetallothionein. Such sequences find use in modulating levels of metalions in plants and plant tissues.

[0014] Expression cassettes comprising the sequences of the inventionare provided. Additionally provided are transformed plant cells, planttissues, and plants.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 depicts a construct used for Agrobacterium-mediatedtransformation utilizing an MT promoter of the invention.

[0016]FIG. 2 depicts root-preferred expression of the MT gene of theinvention, relative to other genes.

[0017]FIG. 3 depicts tissue distribution of expression of the MT gene ofthe invention relative to a second MT gene.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The invention relates to compositions and methods drawn to plantmetallothionein (MT) genes and methods of their use. The compositionscomprise nucleotide sequences for the promoter region of the MT gene ofthe invention, as well as the nucleotide sequences for the expressedregions of the gene.

[0019] The MT promoter sequences of the present invention includenucleotide constructs that allow initiation of transcription in atissue-preferred, more particularly in a root-preferred manner. Suchconstructs of the invention comprise regulated transcription initiationregions associated with plant developmental regulation. Thus, thecompositions of the present invention comprise novel plant promoternucleotide sequences, particularly root-preferred promoter sequences forthe MT gene, more particularly a maize MT promoter sequence. Thesequence for the maize MT promoter region is set forth in SEQ ID NO:1.

[0020] Also provided are compositions and methods for expressing MTgenes in plants, plant cells, and specific plant tissues. Suchcompositions are nucleic acids and proteins relating to MT or MT-likegenes in plants. More particularly, nucleotide sequences encoding maizeMT and the amino acid sequences for the proteins encoded thereby aredisclosed. The MT gene encodes a protein involved in binding andsequestering metal ions. The MT protein contains known Cys-rich motifs.The maize MT gene is abundantly expressed in maize root tissue.Nucleotide sequences for the expressed region of the maize MT genecomprising the MT coding sequences are set forth in SEQ ID NO:2. Themaize MT polypeptide sequence is set forth in SEQ ID NO: 3.

[0021] Compositions of the invention include the nucleotide sequencesfor the native MT promoter and expressed regions, the MT amino acidsequences, as well as fragments and variants thereof. The nucleotidesequences for the expressed region of the MT gene or correspondingantisense sequences find use in modulating the expression ofmetallothionein in a plant or plant cell. That is, the coding sequencesare used to increase the expression while antisense sequences are usedto decrease expression. The promoter sequences of the invention areuseful for expressing sequences in a tissue-preferred, particularly aroot-preferred manner. The sequences of the invention also find use inthe construction of expression vectors for subsequent transformationinto plants of interest, as probes for the isolation of other MT-likegenes, as molecular markers, and the like.

[0022] In particular, the present invention provides for isolatednucleic acid molecules comprising the nucleotide sequences set forth inSEQ ID NOs:1 and 2, isolated nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequences shown in SEQ IDNO:3, or the nucleotide sequences encoding the DNA sequences depositedin a bacterial host as Patent Deposit Nos. 207085 and 207086. Furtherprovided are polypeptides having an amino acid sequence encoded by anucleic acid molecule described herein, for example those set forth inSEQ ID NO: 2, those deposited as Patent Deposit NO. 207086, andfragments and variants thereof.

[0023] Plasmids containing the nucleotide sequences of the inventionwere deposited with the Patent Depository of the American Type CultureCollection (ATCC), Manassas, Va., on Feb. 2, 1999 and assigned PatentDeposit NOs. 207085 and 207086. These deposits will be maintained underthe terms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Thesedeposits were made merely as a convenience for those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112.

[0024] The invention encompasses isolated or substantially purifiednucleic acid or protein compositions. An “isolated” or “purified”nucleic acid molecule or protein, or biologically active portionthereof, is substantially free of other cellular material, or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.Preferably, an “isolated” nucleic acid is free of sequences (preferablyprotein encoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. Aprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, 5%, (bydry weight) of contaminating protein. When the protein of the inventionor biologically active portion thereof is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals. The MT promoter sequences of the invention may be isolatedfrom the 5′ untranslated region flanking their respective transcriptioninitiation sites. Methods for isolation of promoter regions are wellknown in the art.

[0025] Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a MT coding sequence may encode protein fragments thatretain the biological activity of the native protein and hence bindmetal ion. Fragments of a MT promoter sequence may retain the biologicalactivity of driving root-preferred expression. Alternatively, fragmentsof a nucleotide sequence that are useful as hybridization probesgenerally do not encode fragment proteins or promoters retainingbiological activity. Thus, fragments of a nucleotide sequence for theexpressed region of the MT gene may range from at least about 27nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length nucleotide sequence of the invention for the expressedregion of the gene. Fragments of a nucleotide sequence for the promoterregion of the MT gene may range from at least about 20 nucleotides,about 50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence of the invention for the promoter region of thegene.

[0026] A fragment of a MT nucleotide sequence that encodes abiologically active portion of a protein of the invention will encode atleast 15, 25, 30, 50 contiguous amino acids, or up to the total numberof amino acids present in a full-length MT protein of the invention (forexample, 79 amino acids for SEQ ID NO: 3). Fragments of a MT nucleotidesequence that are useful as hybridization probes or PCR primersgenerally need not encode a biologically active portion of a MT protein.

[0027] Thus, a fragment of a MT nucleotide sequence may encode abiologically active portion of a MT protein, MT promoter, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a MT proteincan be prepared by isolating a portion of the nucleotide sequences ofthe invention for the expressed region of the MT gene, expressing theencoded portion of the protein (e.g., by recombinant expression invitro), and assessing the activity of the encoded portion of the MTprotein. Nucleic acid molecules that are fragments of a nucleotidesequence for the expressed region of the MT gene comprise at least 27,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or up tothe number of nucleotides present in a full-length MT sequence disclosedherein (for example, 612 nucleotides for SEQ ID NO: 2.

[0028] A biologically active portion of a MT promoter can be prepared byisolating a portion of the MT promoter sequence of the invention, andassessing the promoter activity of the portion. Nucleic acid moleculesthat are fragments of a MT promoter nucleotide sequence comprise atleast about 16, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700 nucleotides, or up to the number of nucleotidespresent in a full-length MT promoter sequence disclosed herein (forexample, 747 nucleotides for SEQ ID NO: 1).

[0029] By “variants” is intended substantially similar sequences. Fornucleotide sequences, naturally occurring variants can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. For MT coding sequences, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the MTpolypeptides of the invention. Thus, for MT coding sequences, variantsinclude naturally occurring allelic variants. Variant nucleotidesequences also include synthetically derived nucleotide sequences, suchas those generated, for example, by using site-directed mutagenesis. ForMT coding sequences, such synthetically derived sequences still encode aMT protein of the invention.

[0030] Generally, variants of a particular nucleotide sequence of thepresent invention will have at least 70%, generally at least 75%, 80%,85%, preferably about 90% to 95% or more, and more preferably about 98%or more sequence identity to that particular nucleotide sequence asdetermined by sequence alignment programs described elsewhere hereinusing default parameters.

[0031] By “variant” protein is intended a protein derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, metal ion binding activity as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native MT protein of theinvention will have at least at least 80%, 85%, preferably about 90% to95% or more, and more preferably about 98% or more sequence identity tothe amino acid sequence for the native protein as determined by sequencealignment programs described elsewhere herein using default parameters.A biologically active variant of a protein of the invention may differfrom that protein by as few as 1-15 amino acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue.

[0032] The proteins of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the MT proteins can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.), herein incorporated by reference.Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be preferred.

[0033] Thus, the genes and nucleotide sequences of the invention includeboth the naturally occurring sequences as well as mutant forms.Likewise, the proteins of the invention encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Such variants will continue to possess the desired metal bindingactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure. See, EP Patent Application Publication No.75,444.

[0034] The deletions, insertions, and substitutions of the proteinsequences encompassed herein are not expected to produce radical changesin the characteristics of the protein. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays. That is, theactivity can be evaluated by assessing metal ion binding to the isolatedprotein, or by assessing accumulation of metal ions in cells expressingthe protein. See, for example, Robinson et al. (1993) Biochem J. 295:1-10, herein incorporated by reference.

[0035] Variant nucleotide sequences and proteins also encompasssequences and proteins derived from a mutagenic and recombinogenicprocedure such as DNA shuffling. With such a procedure, one or moredifferent MT nucleotide sequences for the promoter or the expressedregion of the gene can be manipulated to create a new MT promoter orprotein possessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the gene of theinvention and other known MT genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedaffinity for metal ions. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

[0036] The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire MT sequences setforth herein or to fragments thereof are encompassed by the presentinvention.

[0037] In a PCR approach, oligonucleotide primers can be designed foruse in PCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

[0038] In hybridization techniques, all or part of a known nucleotidesequence is used as a probe that selectively hybridizes to othercorresponding nucleotide sequences present in a population of clonedgenomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the MT sequences of the invention. Methods for preparation of probesfor hybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

[0039] For example, the entire MT promoter sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding MT sequences and messengerRNAs. To achieve specific hybridization under a variety of conditions,such probes include sequences that are unique among MT sequences and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such probes may be used toamplify corresponding MT sequences from a chosen plant by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism, or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

[0040] Hybridization of such sequences may be carried out understringent conditions. By “stringent conditions” or “stringenthybridization conditions” is intended conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

[0041] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Duration of hybridizationis generally less than about 24 hours, usually about 4 to about 12hours.

[0042] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6(log M)+0.41(%GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, %GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0043] Thus, isolated sequences that have root-preferred promoteractivity or encode for a MT protein and which hybridize under stringentconditions to the MT sequences disclosed herein, or to fragmentsthereof, are encompassed by the present invention. Such sequences willbe at least 70% homologous, and even about 75%, 80%, 85%, 90%, 95% to98% homologous or more with the disclosed sequences. That is, thesequence identity of sequences may range, sharing at least 70%, and evenabout 75%, 80%, 85%, 90%, 95% to 98% or more sequence identity.

[0044] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

[0045] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0046] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

[0047] Methods of alignment of sequences for comparison are well knownin the art. Thus, the determination of percent sequence identity betweenany two sequences can be accomplished using a mathematical algorithm.Preferred, non-limiting examples of such mathematical algorithms are thealgorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homologyalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453; the search-for-similarity-method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlinand Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0048] Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

[0049] Unless otherwise stated, sequence identity/similarity valuesprovided herein refer to the value obtained using GAP version 10 usingthe following parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

[0050] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0051] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0052] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference tothe residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

[0053] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

[0054] (e)(i) The term “substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70% sequence identity, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0055] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C. lower than the T_(m), depending upon the desired degree ofstringency as otherwise qualified herein. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

[0056] (e)(ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%sequence identity to a reference sequence, preferably 80%, morepreferably 85%, most preferably at least 90% or 95% sequence identity tothe reference sequence over a specified comparison window. Preferably,optimal alignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

[0057] The present invention may be used for transformation of any plantspecies, including, but not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

[0058] Heterologous coding sequences expressed by the promoters of theinvention may be used for varying the phenotype of a plant. Variouschanges in phenotype are of interest including modifying expression of agene in a plant root, altering a plant's pathogen or insect defensemechanism, increasing the plants tolerance to herbicides in a plant,altering root development to respond to environmental stress, and thelike. These results can be achieved by providing expression ofheterologous or increased expression of endogenous products in plants.

[0059] Alternatively, the results can be achieved by providing for areduction of expression of one or more endogenous products, particularlyenzymes, transporters, or cofactors, or affecting nutrient uptake in theplant. These changes result in a change in phenotype of the transformedplant.

[0060] General categories of genes of interest for the present inventioninclude, for example, those genes involved in information, such as Zincfingers, those involved in communication, such as kinases, and thoseinvolved in housekeeping, such as heat shock proteins. More specificcategories of transgenes, for example, include genes encoding importanttraits for agronomics, insect resistance, disease resistance, andherbicide resistance. It is recognized that any gene of interest can beoperably linked to the promoter of the invention and expressed in plantroots.

[0061] Insect resistance genes may encode resistance to pests that havegreat yield drag such as rootworm, cutworm, European Corn Borer, and thelike. Such genes include, for example, Bacillus thuringiensis toxicprotein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514;5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); lectins(Van Damme et al. (1994) Plant Mol. Biol. 24:825); and the like.

[0062] Genes encoding disease resistance traits include detoxificationgenes, such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence(avr) and disease resistance (R) genes (Jones et al. (1994) Science266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al.(1994) Cell 78:1089); and the like.

[0063] Herbicide resistance traits may include genes coding forresistance to herbicides that act to inhibit the action of acetolactatesynthase (ALS), in particular the sulfonylurea-type herbicides (e.g.,the acetolactate synthase (ALS) gene containing mutations leading tosuch resistance, in particular the S4 and/or Hra mutations), genescoding for resistance to herbicides that act to inhibit action ofglutamine synthase, such as phosphinothricin or basta (e.g., the bargene), or other such genes known in the art. The bar gene encodesresistance to the herbicide basta, the nptII gene encodes resistance tothe antibiotics kanamycin and geneticin, and the ALS-gene mutants encoderesistance to the herbicide chlorsulfuron.

[0064] Exogenous products include plant enzymes and products as well asthose from other sources including prokaryotes and other eukaryotes.Such products include enzymes, cofactors, hormones, and the like.

[0065] Examples of other applicable genes and their associated phenotypeinclude the gene which encodes viral coat protein and/or RNA, or otherviral or plant genes that confer viral resistance; genes that conferfungal resistance; genes that promote yield improvement; and genes thatprovide for resistance to stress, such as dehydration resulting fromheat and salinity, toxic metal or trace elements, or the like.

[0066] As noted, the heterologous nucleotide sequence operably linked tothe MT promoters disclosed herein may be an antisense sequence for atargeted gene. Thus the promoter sequences disclosed herein may beoperably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant root.

[0067] By “promoter” or “transcriptional initiation region” is intendeda regulatory region of DNA usually comprising a TATA box capable ofdirecting RNA polymerase II to initiate RNA synthesis at the appropriatetranscription initiation site for a particular coding sequence. Apromoter may additionally comprise other recognition sequences generallypositioned upstream or 5′ to the TATA box, referred to as upstreampromoter elements, which influence the transcription initiation rate. Itis recognized that having identified the nucleotide sequences for thepromoter regions disclosed herein, it is within the state of the art toisolate and identify further regulatory elements in the 5′ untranslatedregion upstream from the particular promoter regions identified herein.Additionally, translational fusions may be provided. Such fusionsinclude portions of the amino acid sequence. Thus the promoter regionsdisclosed herein are generally further defined by comprising upstreamregulatory elements such as, those responsible for tissue and temporalexpression of the coding sequence, enhancers and the like. In the samemanner, the promoter elements, which enable expression in the desiredtissue such as the root, can be identified isolated and used with othercore promoters to confer root-preferred expression. In this aspect ofthe invention, by “core promoter” is intended a promoter withoutpromoter elements.

[0068] The compositions of the invention include promoter elementsidentified in the MT promoter sequences of the invention. These elementsinclude, but are not limited to the promoter elements having thenucleotide sequences TATGAGATGA; CGATCGACAA; GGCACAAGA; GATATAGAT; thenucleotide sequence set forth in SEQ ID NO:9; AGAGCACGC; AGT TCTG;AGCTGTA; AT AGATTAC. These elements correspond to nucleotides 39-48,179-188, 295-303, 305-313, 422-436, 444-452, 520-526, 616-622, and671-679 of the promoter sequence set forth in SEQ ID NO:1, respectively.It is determined that these elements contribute to the root-preferredexpression of MT.

[0069] It is further determined that, these promoter elements, whenintroduced into minimal or constitutive promoters, direct root preferredexpression in a plant. The invention encompasses promoters that driveroot-preferred expression and comprise at least one copy of at least oneroot-preferred promoter element selected from the group consisting ofTATGAGATGA; CGATCGACAA; GGCACAAGA; GATATAGAT; the nucleotide sequenceset forth in SEQ ID NO:9; AGAGCACGC; AGT TCTG; AGCTGTA; AT AGATTAC.

[0070] In a preferred embodiment, the root-preferred promoter of thepresent invention comprises at least one copy of at least one promoterelement selected among promoter elements having the nucleotide sequenceCGATCGACAA; the nucleotide sequence set forth in SEQ ID NO:9; AGAGCACGC;AGTTCTG; AGCTGTA; ATAGATTAC. In a more preferred embodiment, theroot-preferred promoter of the present invention comprises at least onecopy of the promoter element having the nucleotide sequence AGAGCACGC.

[0071] The regulatory sequences of the present invention, when operablylinked to a heterologous nucleotide sequence of interest and insertedinto a transformation vector, drive root-preferred expression of theheterologous nucleotide sequence in the root stably transformed withthis vector. By “root-preferred” is intended that expression of theheterologous nucleotide sequence is most abundant in the root includingat least one of root cap, apical meristem, protoderm, ground meristem,procambium, endodermis, cortex, vascular cortex, epidermis, and thelike. While some level of expression of the heterologous nucleotidesequence may occur in other plant tissue types, expression occurs mostabundantly in the root including primary, lateral and adventitiousroots.

[0072] By “heterologous nucleotide sequence” is intended a sequence thatis not naturally occurring with the promoter sequence. While thisnucleotide sequence is heterologous to the promoter sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.

[0073] The isolated promoter sequences of the present invention can bemodified to provide for a range of expression levels of the heterologousnucleotide sequence. Thus, less than the entire promoter regions may beutilized and the ability to drive expression of the coding sequenceretained. However, it is recognized that expression levels of the mRNAmay be decreased with deletions of portions of the promoter sequences.Generally, at least about 20 nucleotides of an isolated promotersequence will be used to drive expression of a nucleotide sequence.

[0074] It is recognized that to increase transcription levels enhancersmay be utilized in combination with the promoter regions of theinvention. Enhancers are nucleotide sequences that act to increase theexpression of a promoter region. Enhancers are known in the art andinclude the SV40 enhancer region, the 35S enhancer element, and thelike.

[0075] Modifications of the isolated promoter sequences of the presentinvention can provide for a range of expression of the heterologousnucleotide sequence. Thus, they may be modified to be weak promoters orstrong promoters. Generally, by “weak promoter” is intended a promoterthat drives expression of a coding sequence at a low level. By “lowlevel” is intended at levels of about 1/10,000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts. Conversely, astrong promoter drives expression of a coding sequence at a high level,or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000transcripts.

[0076] It is recognized that the promoters of the invention may be usedwith their native MT coding sequences to increase or decreaseexpression, thereby resulting in a change in phenotype of thetransformed plant. This phenotypic change could further effect anincrease or decrease in levels of metal ions in tissues of thetransformed plant.

[0077] The MT coding sequences of the present invention can be used withpromoters known in the art. Such sequences find use in modulating uptakeof metal ions. Such metals include cadmium, zinc, copper, mercury, gold,silver, cobalt, nickel, bismuth, and the like.

[0078] The MT coding sequences may additionally be used to regulate geneexpression, particularly by coordination of zinc binding and expressionduring development. The MT proteins are implicated in the sequestrationof copper in roots and play a role in metabolism of metal ions.Additionally, the MT polypeptides may play an antioxidant role as DNAstrand-breakage, induced by oxidative stress, is reduced in the presenceof elevated metallothionein levels. Thus, antisense constructscorresponding to MT coding sequences or MT antibodies may find use inincreasing strand breakage for enhancement of transformation insertionevents.

[0079] It is recognized that with the MT coding sequences, antisenseconstructions, complementary to at least a portion of the messenger RNA(mRNA) for the MT sequences can be constructed. It is also recognizedthat with the MT promoter sequences, antisense constructions,complementary to at least a portion of the messenger RNA (mRNA) for anytargeted sequence(s) can be constructed. Antisense nucleotides areconstructed to hybridize with the corresponding mRNA. Modifications ofthe antisense sequences may be made as long as the sequences hybridizeto and interfere with expression of the corresponding mRNA. In thismanner, antisense constructions having 70%, preferably 80%, morepreferably 85% sequence identity to the corresponding antisensedsequences may be used. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, or greater may be used.

[0080] The nucleotide sequences of the present invention may also beused in the sense orientation to suppress the expression of endogenousgenes in plants. Methods for suppressing gene expression in plants usingnucleotide sequences in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, preferably greater than about 65% sequenceidentity, more preferably greater than about 85% sequence identity, mostpreferably greater than about 95% sequence identity. See, U.S. Pat. Nos.5,283,184 and 5,034,323; herein incorporated by reference.

[0081] The coding sequences of the invention are useful for transformingplants and modulating levels of metal ions in a plant. By “modulatinglevels of metal ions” is intended an increase or a decrease in theamount of the ionic form of a metal. In this aspect of the invention, itis envisioned that the binding of the MT protein expressed by thesequences of the invention to a metal ion of interest will render ametal-ligand protein(MT) complex, thereby decreasing the amount of theionic form of the metal. Alternatively, antisense sequences to thecoding sequences of the invention may be used to increase the level ofmetal ions of interest. The metal ions are cations, more particularlydivalent cations, even more particularly Cu⁺⁺.

[0082] Generally, decreased metal ion content in plant tissue may reducethe toxicity associated with excessive amounts of metal ions in thetissue. Toward this end, the sequences of the invention may be utilizedin expression cassettes or DNA constructs with tissue-preferredpromoters including but not limited to seed-specific promoters (thosepromoters active during seed development), as well as seed-germinatingpromoters (those promoters active during seed germination). Suchseed-specific promoters include Cim1 (cytokinin-induced message); cZ19B1(maize 19KDa zein); mi1ps (myo-inositol-1-phosphate synthase); celA(cellulose synthase); end1 (Hordeum verlgase mRNA clone END1); alphaamylase; and imp3 (myo-inositol monophosphate-3). For dicots, particularpromoters include phaseolin, napin, β-conglycinin, soybean lectin, andthe like. For monocots, particular promoters include maize 15Kd zein,22KD zein, 27kD zein, waxy, shrunken 1, shrunken 2, globulin 1, etc.

[0083] For expression of the nucleotide sequences of the inventioncomprising the MT coding sequences, constitutive or tissue-preferredpromoters may be utilized. Constitutive promoters would provide aconstant supply of MT protein throughout the plant. Such constitutivepromoters include, for example, the core promoter of the Rsyn7(copending U.S. patent application Ser. No. 08/661,601), the core CaMV35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen etal. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992)Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl.Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. patent application Ser. No. 08/409,297), and the like.Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; and 5,608,142.

[0084] The utilization of tissue-preferred promoters with the codingsequences of the invention would increase or decrease the availabilityof a metal ion of interest in specific tissues of the plant. Forexample, leaf-specific promoters may be utilized. Such tissue-preferredpromoters include, Yamamoto et al. (1997) Plant J. 12(2):255-265;Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al.(1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) TransgenicRes. 6(2):157-168; Rinehart et al. (1996) Plant Physiol.112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535;Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al.(1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. CellDiffer. 20:181-196; Orozco et al. (1993) Plant Mol. Biol.23(6):1129-11-38; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505.

[0085] In particular, one tissue-preferred promoter of interest includesroot-preferred promoters. The utilization of such promoters wouldprovide a mechanism for modulating the level of metal ions in the root,and influence the uptake of the metal ions by the root. Thus, theinvention encompasses increasing root uptake of metal ions by expressionof the coding sequences of the invention; and decreasing root uptake ofmetal ions by transforming roots with corresponding antisense sequences.Root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster Het al. (1995) Plant Mol Biol. 29(4):759-772); and rolB promoter (Capanaet al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos.5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179 and the MTpromoter sequences disclosed herein.

[0086] The nucleotide sequences disclosed in the present invention, aswell as variants and fragments thereof, are useful in the geneticmanipulation of any plant. The MT promoter sequences are useful in thisaspect when operably linked with a heterologous nucleotide sequencewhose expression is to be controlled to achieve a desired phenotypicresponse. By “operably linked” is intended the transcription ortranslation of the heterologous nucleotide sequence is under theinfluence of the promoter sequence. In this manner, the nucleotidesequences for the promoters of the invention may be provided inexpression cassettes along with heterologous nucleotide sequences ofinterest for expression in the plant of interest, more particularly inthe root of the plant.

[0087] Such expression cassettes will comprise a transcriptionalinitiation region comprising one of the promoter nucleotide sequences ofthe present invention, or variants or fragments thereof, operably linkedto the heterologous nucleotide sequence whose expression is to becontrolled by the root-preferred promoters disclosed herein. Such anexpression cassette is provided with a plurality of restriction sitesfor insertion of the nucleotide sequence to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

[0088] The MT coding sequences of the invention are also provided inexpression cassettes for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked tothe sequence of interest.

[0089] The sequences of the invention can be introduced into any plant.The sequences to be introduced may be used in expression cassettes forexpression in any plant of interest where expression in the plant isnecessary for transcription.

[0090] Plants of interest include, but are not limited to corn (Zeamays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), oats, barley, vegetables, ornamentals, andconifers. Preferably plants include corn, soybean, sunflower, safflower,Brassica, wheat, barley, rye, alfalfa, and sorghum.

[0091] The expression cassette comprising the sequences of the inventionwill include in the 5′-to-3′ direction of transcription, atranscriptional and translational initiation region, a nucleotidesequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region comprising one of thepromoter nucleotide sequences of the present invention, may be nativewith the DNA sequence of interest, or may be derived from anothersource. Convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. 1989) Nucleic Acids Res.17:7891-7903; Joshi heterologous et al. (1987) Nucleic Acid Res.15:9627-9639.

[0092] The expression cassette comprising the sequences of the presentinvention may also contain at least one additional nucleotide sequencefor a gene to be cotransformed into the organism. Alternatively, theadditional sequence(s) can be provided on another expression cassette.

[0093] Where appropriate, the nucleotide sequences whose expression isto be under the control of the root-preferred promoter sequence of thepresent invention and any additional nucleotide sequence(s) may beoptimized for increased expression in the transformed plant. That is,these nucleotide sequences can be synthesized using plant preferredcodons for improved expression. Methods are available in the art forsynthesizing plant-preferred nucleotide sequences. See, for example,U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) NucleicAcids Res. 17:477-498, herein incorporated by reference.

[0094] Additional sequence modifications are known to enhance geneexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe heterologous nucleotide sequence may be adjusted to levels averagefor a given cellular host, as calculated by reference to known genesexpressed in the host cell. When possible, the sequence is modified toavoid predicted hairpin secondary mRNA structures.

[0095] The expression cassettes may additionally contain 5′ leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Nat. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example,TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader(Maize Dwarf Mosaic Virus) (Virology 154:9-20); human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991) Nature353:90-94); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);tobacco mosaic virus leader (TMV) (Gallie et al. (1989) MolecularBiology of RNA, pages 237-256); and maize chlorotic mottle virus leader(MCMV) (Lommel et al. (1991) Virology 81:382-385). See also Della-Cioppaet al. (1987) Plant Physiology 84:965-968. Other methods known toenhance translation and/or mRNA stability can also be utilized, forexample, introns, and the like.

[0096] In preparing the expression cassette, the various DNA fragmentsmay be manipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, for example,transitions and transversions, may be involved.

[0097] Reporter genes or selectable marker genes may be included in theexpression cassettes. Examples of suitable reporter genes known in theart can be found in, for example, Jefferson et al. (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer AcademicPublishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737;Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques19:650-655; and Chiu et al. (1996) Current Biology 6:325-330.

[0098] Selectable marker genes for selection of transformed cells ortissues can include genes that confer antibiotic resistance orresistance to herbicides. Examples of suitable selectable marker genesinclude, but are not limited to, genes encoding resistance tochloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992);methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213; Meijeret al. (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al.(1985) Plant Mol. Biol. 5:103-108; Zhijian et al. (1995) Plant Science108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet.210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) TransgenicRes. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker et al. (1988) Science 242:419-423);glyphosate (Shaw et al. (1986) Science 233:478-481); phosphinothricin(DeBlock et al. (1987) EMBO J. 6:2513-2518).

[0099] Other genes that could serve utility in the recovery oftransgenic events but might not be required in the final product wouldinclude, but are not limited to, examples such as GUS (β-glucuronidase;Jefferson (1987) Plant Mol. Biol. Rep. 5:387), GFP (green florescenceprotein; Chalfie et al. (1994) Science 263:802), luciferase (Riggs etal. (1987) Nucleic Acids Res. 15(19):8115 and Luehrsen et al. (1992)Methods Enzymol. 216:397-414) and the maize genes encoding foranthocyanin production (Ludwig et al. (1990) Science 247:449).

[0100] The expression cassette comprising the MT promoter or codingsequence of the present invention can be used to transform any plant. Inthis manner, genetically modified plants, plant cells, plant tissue,seed, root, and the like can be obtained.

[0101] Transformation protocols as well as protocols for introducingnucleotide sequences into plants may vary depending on the type of plantor plant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plant cellsand subsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No.5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S.Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into IntactPlant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, andOrgan Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buisinget al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

[0102] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL

[0103] The maize MT gene was isolated from maize plants. The nucleotidesequences for the maize MT promoter region is set forth in SEQ ID NO:1,and the nucleotide sequence for the expressed region of the gene is setforth in SEQ ID NO:2. The sequence for the expressed region of the MTgene set forth in SEQ ID NO:2 consists of the 5′ untranslated region (5′UTR, nucleotides 1-68), the maize MT polypeptide encoding region(nucleotides 69-308), and the 3′ untranslated region (ncleotides309-612).

EXAMPLE 1

[0104] Isolation of MT Promoter Sequences

[0105] The procedure for promoter isolation is described in the UserManual for the Genome Walker kit sold by Clontech Laboratories, Inc.,Palo Alto, Calif. Genomic DNA from maize line V3-4 A63 was prepared bygrinding 10-day-old seedling leaves in liquid nitrogen, and the DNAprepared as described by Chen and Dellaporta (1994) in The MaizeHandbook, ed. Freeling and Walbot (Springer-Verlag, Berlin) with a fewminor modifications. Precipitated DNA was recovered using an inoculationloop and transferred to a 1.5 ml eppendorf tube containing 500 μl of TE(10 mM Tris pH 8.0, 1 mM EDTA). The DNA was allowed to dissolve at roomtemperature for 15 minutes, phenol extracted and 2-propanol precipitatedin 700 μl. The precipitate was recovered and washed with 70% ethanol.The DNA was then placed in a clean 1.5 ml eppendorf tube to air dry andresuspended in 200 μl of TE. RNase A was added to 10 μg/ml and themixture was incubated at 37° C. for several hours. The DNA was thenextracted once with phenol-chloroform, then chloroform, then ethanolprecipitated and resuspended in TE. The DNA was then used as describedin the Genome Walker User Manual (Clontech PT3042-1 version PR68687).Briefly, the DNA was digested separately with restriction enzymes DraI,EcoRV, PvuII, ScaI, and StuI, all blunt-end cutters. The DNA wasextracted with phenol, then chloroform, then ethanol precipitated. TheGenome Walker adapters were ligated onto the ends of the restricted DNA.The resulting DNA is referred to as DL1-DL5, respectively.

[0106] For isolation of specific promoter regions, three nonoverlappinggene-specific primers (21-26 bp in length) were designed from the 5′ endof the maize genes identified from sequence databases. The primers weredesigned to amplify the region upstream of the coding sequence, i.e. the5′ untranslated region and promoter of the chosen gene. The sequence ofthe primers are given below. The first round of PCR was performed oneach DNA sample (DL1-5) with Clontech primer AP1 (sequence5′-gtaatacgactcactatagggc-3′) and the gene-specific primer (gsp)1 withthe sequences shown in SEQ ID NO:4.

[0107] PCR was performed in a model PTC-100 thermal cycler withHotBonnet from M J Research (Watertown, Mass.) using reagents suppliedwith the Genome Walker kit and the Advantage Genomic PCR Kit (ClontechK1906-y). The following cycle parameters were used: 7 cycles of 94° C.for 2 seconds, then 72° C. for 3 minutes, followed by 37 cycles of 94°C. for 2 seconds and 67° C. for 3 minutes. Finally, the samples wereheld at 67° C. for 4 minutes and then at 4° C. until further analysis.

[0108] As described in the User Manual, the DNA from the first round ofPCR was then diluted and used as a template in a second round of PCRusing the Clontech AP2 primer (sequence 5′-actatagggcacgcgtggt-3′) andgene-specific primer (gsp)2 with the sequences shown in SEQ ID NO:5. Thecycle parameters for the second round were 5 cycles of 94° C. for 2seconds, then 72° C. for 3 minutes, followed by 20 more cycles of 94° C.for 2 seconds, then 67° C. for 3 minutes. Finally, the samples were heldat 67° C. for 4 minutes and then held at 4° C.

[0109] The DNA from the second round of PCR was then diluted and used asa template in a third round of PCR using the Clontech AP2 primer andgene-specific primer (gsp)3 with the sequence shown in SEQ ID NO:6. Thecycle parameters for the third round of PCR were 93° C. for 1.5 minutes,followed by 35 cycles of 93° C. for 30 seconds, then 58° C. for 30seconds, then 72° C. for 3 minutes. Finally, the samples were held at72° C. for 10 minutes and then held at 4° C. Approximately 10 μl of eachreaction were run on a 0.7% agarose gel, transferred to a solid supportand hybridized with a labeled probe designated CRVAC17. Bands that boundto the CRVAC17 probe (usually 500 bp or larger) were excised out of aseparate 0.7% agarose gel, purified with the PCR Cleanup Kit (Promega,Madison, Wis.) and cloned into the Zero Blunt vector, pCR-Blunt(Invitrogen, San Diego, Calif.). Clones were sequenced for verification.

[0110] A clone designated rmp5B corresponding to the promoter sequenceset forth in SEQ ID NO:1 upstream of nucleotides 1-65 of SEQ ID NO:2 wasdeposited as ATCC Patent Deposit NO: 207085, and a cDNA corresponding toSEQ ID NO:2 and the CRVAC17 probe was deposited as ATCC deposit NO:207086; as indicated above.

EXAMPLE 2

[0111] Transformation and Regeneration of Transgenic Plants

[0112] Immature maize embryos from greenhouse donor plants are bombardedwith a plasmid containing a gene of interest operably linked to a MTpromoter of the invention, or a plasmid comprising the MT codingsequences of the invention operably linked to a desired promoter (e.g. aubiquitin promoter), plus a plasmid containing the selectable markergene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistanceto the herbicide Bialaphos. Transformation is performed as follows.Media recipes follow below.

[0113] Preparation of Target Tissue

[0114] The ears are surface sterilized in 30% Chlorox bleach plus 0.5%Micro detergent for 20 minutes, and rinsed two times with sterile water.The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

[0115] Preparation of DNA

[0116] A plasmid vector comprising a gene of interest operably linked toa MT promoter of the invention, or a plasmid comprising the MT codingsequences of the invention operably linked to a desired promoter (e.g. aubiquitin promoter), is made. This plasmid DNA plus plasmid DNAcontaining a PAT selectable marker is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl₂ precipitation procedure asfollows:

[0117] 100 μl prepared tungsten particles in water

[0118] 10 μl (1 μg) DNA in TrisEDTA buffer (1 μg total)

[0119] 100 μl 2.5 M CaCl₂

[0120] 10 μl 0.1 M spermidine

[0121] Each reagent is added sequentially to the tungsten particlesuspension, while maintained on the multitube vortexer. The finalmixture is sonicated briefly and allowed to incubate under constantvortexing for 10 minutes. After the precipitation period, the tubes arecentrifuged briefly, liquid removed, washed with 500 ml 100% ethanol,and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl100% ethanol is added to the final tungsten particle pellet. Forparticle gun bombardment, the tungsten/DNA particles are brieflysonicated and 10 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

[0122] Particle Gun Treatment

[0123] The sample plates are bombarded at level #4 in particle gun#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with atotal of ten aliquots taken from each tube of prepared particles/DNA.

[0124] Subsequent Treatment

[0125] Following bombardment, the embryos are kept on 560Y medium for 2days, then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for root-preferred activity of the geneof interest, or for altered metal ion levels.

[0126] Bombardment and Culture Media

[0127] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added aftersterilizing the medium and cooling to room temperature).

[0128] Plant regeneration medium (288J) comprises 4.3 g/l MS salts(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 gnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-I H₂O) (Murashige andSkoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/lzeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought tovolume with polished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite(added after bringing to volume with D-I H₂O); and 1.0 mg/l indoleaceticacid and 3.0 mg/l bialaphos (added after sterilizing the medium andcooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MSsalts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/lnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-I H₂O), 0.1 g/lmyo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-IH₂O after adjusting pH to 5.6); and 6 g/l bacto-agar (added afterbringing to volume with polished D-I H₂O), sterilized and cooled to 60°C.

EXAMPLE 3

[0129] Expression Data Using the Promoter Sequences of the Invention

[0130] Nine μg of a transformation vector comprising the β-glucuronidase(GUS) operably linked to a promoter of the invention, plus 1 μg of aplasmid comprising the luciferase reporter gene operably linked to aubiquitin promoter (ubi::LUC) to act as a standard control isprecipitated onto tungsten particles and bombarded onto maize embryos asdescribed above. Shoots and roots are harvested separately and measuredfor GUS activity.

EXAMPLE 4

[0131] Transformation and Regeneration of Transgenic Plants UsingAgrobacterium Mediated Transformation

[0132] For Agrobacterium-mediated transformation of maize with anucleotide sequence of interest operably linked to a MT promoter of theinvention, or with the MT coding sequences of the invention, preferablythe method of Zhao is employed (U.S. Pat. No. 5,981,840), the contentsof which are hereby incorporated by reference. In this method, immatureembryos are isolated from maize and the embryos contacted with asuspension of Agrobacterium, where the bacteria are capable oftransferring the gene of interest, or the MT coding sequences, to atleast one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos are preferablyimmersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). Preferably the immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants.

EXAMPLE 5

[0133] Expression Analysis of the Metallothionein Promoter

[0134] Transient expression assays in immature maize embryos:

[0135] The rmp5b genomic fragment containing the MT promoter and part ofthe 5′ UTR was ligated into an expression vector containingβ-glucuronidase (GUS) coding sequences. Such a vector is shown in FIG.1, Plasmid M. The vector was constructed for Agrobacterium-mediatedtransformation as described in Example 4 above. The vector contained aRight border region(RB); an expression cassette comprising the MTpromoter (MET PRO), the MT 5′ UTR sequences (MT 5UTR), GUS exon1, thepotato ST-LS1 intron2, GUS exon 2, and the potato proteinase inhibitortermination region (PINII TERM) for GUS expression; an expressioncassette comprising the CaMV 35 S promoter (CAMV35S PRO), PAT selectionmarker, and the CaMV 35 S termination region (CAMV 35S TERM) for planttransformant selection; and a left border (LB).

[0136] Immature embryos were then transformed with plasmid M viaAgrobacterium-mediated transformation as described in Example 4 above togenerate stable maize transformants. A control plasmid, identical toplamid M except containing the constitutive ubiquitin promoter andenhancer region (ubiquitin exon 1 and part of ubiquitin intron 1) ratherthan the MT promoter and UTR, was used as a positive control.

[0137] Staining of Immature Embryos for GUS Activity

[0138] GUS staining of GS3 immature embryos co-cultivating withAgrobacterium containing either the control plasmid or plasmid Mconsisted of placing embryos into the wells of a 48-well platecontaining 0.5ml of x-gluc solution (0.5 g x-gluc dissolved in 20 mlDMSO and 0.16 g K₄Fe(CN)₆ added separately to 1 L of 0.1M Na₂HPO₄, pH7,0.01M EDTA, 0.1% TritonX-100, 10% Methanol). Each plate containedimmature embryos from either individual ears or a pool of 2-3 ears. Theplates were sealed and incubated at 37° C. for 18 h after which thex-gluc solution was replaced with 0.5 ml of 70% ethanol. The results aresummarized in Table I. TABLE I Total No. of expressing GS3 embryos HoursAfter Co-cultivation Plasmid M Control 24 0 of 8  8 of 8 48 3 of 48 68of 68 120 21 of 48  68 of 68

[0139] These results indicated that rmp5B genomic fragment comprisingthe metallothionein promoter was able to direct expression of the GUScoding region. Transgenic plants are regenerated to determine thestrength, as well as root-specificity of the promoter.

EXAMPLE 6

[0140] Expression of the MT Gene in Maize

[0141] Expression of multiple genes (ESTs) in leaves (the 6 ^(th) leaf),stems (stalk), emerging crown roots (top nodal roots), adventitiousroots, and corn rootworm (CRW)-eaten adventitious roots of V6 maizeplants were compared, utilizing the GeneChip® microarray technology. SeeLipshutz et al. (1999) Nature Genetics Supplement 21: 20-24; Li et al.(1999) Developmental Biology 211: 64-76. Briefly, using known methods,cDNA was synthesized from polyA+RNA isolated from the indicated tissues.Biotin-labeled cRNA was synthesized by in-vitro transcription of thiscDNA using biotin-conjugated ribonucleotides. The cRNA was fragmented,hybridized to a customized GeneChip® array of Zea mays, washed, andstained with streptavidin (R-phycoerythrin conjugate) using theAffymetrix Inc. GeneChip Fluidics station. The Hewlett-Packard G2500AGene Array Scanner and Affymetrix GeneChip Analysis software were usedto analyze the results.

[0142] The developmental stage, V6, was selected because Western cornrootworm WCRW feed on maize roots between the stages of V4 and V8. Thus,a promoter active during these stages would be ideal to control theexpression of a CRW insecticidal gene. V6 is defined by the maturationof the collar of the sixth leaf of the plant. CRW-eaten adventitiousroots were generated by infesting each pot with 50 WCRW eggs. Thisresulted in roots that were damaged and scarred, but not decimated.

[0143] Analysis of the GeneChip results indicated the gene for ESTCVRAQ62 (sequence set forth in SEQ ID NO:7), which belongs to the ESTfamily corresponding to the nucleotide sequences for the expressedregion of the maize MT gene (SEQ ID NO:2), was expressed in V6adventitious roots at levels >250-fold higher than in V6 leaves. Thesame gene was also expressed >100-fold higher in V6 roots compared to V6stalks. Comparing V6 adventitious roots to V6 crown roots showed onlyabout a 2-fold greater level of expression in adventitious roots. A lessthan 2-fold difference was detected between adventitious roots andWCRW-eaten roots. These results indicate that the metallothionein geneof the present invention is highly expressed in root tissue, but not inleaf or stalk tissue. These data also indicate a high level ofexpression in emerging crown roots and in WCRW-eaten roots as there wasnot greater than a 3-fold difference in expression between adventitiousroots, crown roots and WCRW-eaten roots. High expression in WCRW-eatenroots is significant because it indicates insect feeding does notdownregulate the promoter.

[0144] A second set of GeneChip microarray experiments were performedessentially as described above, and representative results are shown inFIG. 2. These results indicate high expression of the MT gene of thepresent invention represented by EST CRVAQ62, relative to a second maizeMT gene represented by the EST CRWBJ81 (sequence set forth in SEQ IDNO:8) and other genes as shown in FIG. 2.

[0145] Northern blot experiments indicating root-preferred expression ofthe MT gene of the invention are not available. Electronic Northernblots showed tissue distribution of the MT gene of the present inventionrepresented by EST CRVAQ62, relative to the second maize MT generepresented by the EST CRWBJ81; the results shown in FIG. 3.

[0146] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0147] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

1 18 1 747 DNA Zea mays promoter (1)...(747) Promoter sequence for maizemetallothionein 1 cgacgggcat ttgcgtagtt gaagcttaca aagttgcata tgagatgagtgccggacatg 60 aagcggataa cgttttaaac tggcaacaat atctagctgt ttcaaattcaggcgtgggaa 120 gctacgccta cgcgccctgg acggcgtgta aagagccagc atcggcatcattgtcaaacg 180 atcgacaagg ccaagaaatt ccaaatatat tattaataaa aaagaaggcacaaattagtt 240 tggtttttta gtatgtgtgg cggaggaaat tttgagaacg aacgtatcaaagaaggcaca 300 agacgatata gattgacgcg gctagaagtt gcagcaagac agtgggtacggtcttatata 360 tcctaataaa taaaaaataa aactatagtg tgtcaaatgt caacaagaggaggaggcagc 420 caaattagca gagggagaca agtagagcac gccttattag cttgcttatttatcgtggtg 480 gtgtacttgt taattactgg cacgcattat caacaacgca gttctggatgtgaatctaga 540 caaacatttg tctaggttcc gcacgtatag tttttttcct cttttttttggggggggggt 600 ggggggggga acggaagctg taataaacgg tactaggaac gaaagcaaccgccgcgcgca 660 tgtttttgca atagattacg gtgaccttga tgcaccaccg cgtgctataaaaaccagtgt 720 ccccgagtct actcatcaac caatcca 747 2 612 DNA Zea mays CDS(69)...(308) Coding sequence for maize metallothionein 2 taactcgaaaccttttcttg tgctctgttc tgtctgtgtg tttccaaagc aaacgaaaga 60 ggtcgagg atgtct tgc agc tgc gga tca agc tgc aac tgc gga tca agc 110 Met Ser Cys SerCys Gly Ser Ser Cys Asn Cys Gly Ser Ser 1 5 10 tgc aag tgc ggc aag atgtac cct gac ctg gag gag aag agc ggc ggg 158 Cys Lys Cys Gly Lys Met TyrPro Asp Leu Glu Glu Lys Ser Gly Gly 15 20 25 30 ggc gct cag gcc agc gccgcc gcc gtc gtc ctc ggc gtt gcc cct gag 206 Gly Ala Gln Ala Ser Ala AlaAla Val Val Leu Gly Val Ala Pro Glu 35 40 45 acg aag aag gcg gcg cag ttcgag gcg gcg ggc gag tcc ggc gag gcc 254 Thr Lys Lys Ala Ala Gln Phe GluAla Ala Gly Glu Ser Gly Glu Ala 50 55 60 gct cac ggc tgc agc tgc ggt gacagc tgc aag tgc agc ccc tgc aac 302 Ala His Gly Cys Ser Cys Gly Asp SerCys Lys Cys Ser Pro Cys Asn 65 70 75 tgc tga tcctgctgcg ttgtttcgtttgcggcatgc atggatgtca cctttttttt 358 Cys * actgtctgct ttgtgcttgtggcgtgtcaa gaataaagga tggagccatc gtctggtctg 418 actctggctc tccgccatgcatgcttggtg tcggttctgt tgtgcttgtg ttggtgcatg 478 taatcgtatg gcatcgttacacaccatgca tctctgatct ctttgcgcca gtgtgtgtga 538 ctatgtccct gtaacgattggctcagtgat tgaatatata tacaatactg ttttactaaa 598 aaaaaaaaaa aaaa 612 3 79PRT Zea mays 3 Met Ser Cys Ser Cys Gly Ser Ser Cys Asn Cys Gly Ser SerCys Lys 1 5 10 15 Cys Gly Lys Met Tyr Pro Asp Leu Glu Glu Lys Ser GlyGly Gly Ala 20 25 30 Gln Ala Ser Ala Ala Ala Val Val Leu Gly Val Ala ProGlu Thr Lys 35 40 45 Lys Ala Ala Gln Phe Glu Ala Ala Gly Glu Ser Gly GluAla Ala His 50 55 60 Gly Cys Ser Cys Gly Asp Ser Cys Lys Cys Ser Pro CysAsn Cys 65 70 75 4 26 DNA Artificial Sequence Gene specific syntheticprimer for MT promoter isolation 4 atcttgccgc acttgcagct tgatcc 26 5 24DNA Artificial Sequence Gene specific primer for MT promoter isolation 5cagttgcagc ttgatccgca gctg 24 6 21 DNA Artificial Sequence Gene specificprimer for MT promoter isolation 6 caggatcctc gacctctttc g 21 7 255 DNAZea mays 7 cttgcaactg cggatcaagc tgcggctgcg gctcaagctg caagtgcggcaagaagtacc 60 ctgacctgga ggagacgagc accgccgcgc aggccaccgt cgtcctcggcgtggccccgg 120 agaagaaggc cgcgcccgag ttcgtcgagg ccgcggcgga gtccggcgaggccgcccacg 180 gctgcagctg cggtggcaac tgcaagtgcg acccctgcaa ctgctgatcacatcgatcga 240 cgaccatgga tatga 255 8 255 DNA Zea mays 8 gtgctctgttctgtctgtgt gtttccaaag caaacgaaag aggtcgagga tgtcttgcag 60 ctgcggatcaagctgcaact gcggatcaag ctgcaagtgc ggcaagatgt accctgacct 120 ggaggagaagagcggcgggg gcgctcaggc cagcgccgcc gccgtcgtcc tcggcgttgc 180 ccctgagacgaagaaggcgg cgcagttcga ggcggcgggc gagtccggcg aggccgctca 240 cggctgcagctgcgg 255 9 15 DNA Zea mays misc_feature (1)...(15) Maize promoterelement 9 aaattagcag aggga 15 10 10 DNA Zea mays misc_feature (1)...(10)Maize promoter element 10 tatgagatga 10 11 10 DNA Zea mays misc_feature(1)...(10) Maize promoter element 11 cgatcgacaa 10 12 9 DNA Zea maysmisc_feature (1)...(9) Maize promoter element 12 ggcacaaga 9 13 9 DNAZea mays misc_feature (1)...(9) Maize promoter element 13 gatatagat 9 149 DNA Zea mays misc_feature (1)...(9) Maize promoter element 14agagcacgc 9 15 7 DNA Zea mays misc_feature (1)...(7) Maize promoterelement 15 agttctg 7 16 7 DNA Zea mays misc_feature (1)...(7) Maizepromoter element 16 agctgta 7 17 9 DNA Zea mays misc_feature (1)...(9)Maize promoter element 17 atagattac 9 18 19 DNA Artificial SequenceOligonucleotide primer 18 actatagggc acgcgtggt 19

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence set forth in SEQ ID NO:3.
 2. An isolated polypeptide comprising25 contiguous amino acids of SEQ ID NO:3 wherein said polypeptide hasmetal binding activity.
 3. The polypeptide of claim 2, wherein saidpolypeptide comprises 30 contiguous amino acids.
 4. The polypeptide ofclaim 3, wherein said polypeptide comprises 50 contiguous amino acids.5. An isolated polypeptide having 70% sequence identity with SEQ ID NO:3wherein said polypeptide has metal binding activity.
 6. The polypeptideof claim 5, wherein said polypeptide has 80% sequence identity with SEQID NO:3.
 7. The polypeptide of claim 6, wherein said polypeptide has 90%sequence identity with SEQ ID NO:3.
 8. The polypeptide of claim 6,wherein said polypeptide has 95% sequence identity with SEQ ID NO:3.